PhD defence by Christa Kanstrup

Picture of cell transport

Elucidation of the structure-function relationship in the Nitrate and Peptide Transporter Family

The Nitrate and Peptide transporter Family (NPF) are reported to have significant roles in nutrient use efficiency vital for agriculture, in transporting phytohormones essential for physiological processes in plants, and in transport of plant specialized metabolites – metabolites that can be both harmful and beneficial to humans. Even though this family is important, it is in many aspects unexplored. Many properties of the mechanism of transport and substrate recognition within the transporters are mysteries. To date, only one NPF transporter – from the model plant Arabidopsis thaliana – has been crystalized, and only in a single conformation. The little information we have of the structure-function relationship of the family comes from mutational studies that often lead to loss-of-function mutations that simply states that the amino acid is important for function, meaning that mechanistic descriptions are lacking. 


In this dissertation, I want to extend our knowledge on amino acid positions important for substrate specificity and transport rate in NPF transporters. The well-conserved and distant related transport family of Proton-coupled Oligopeptide Transporters (POTs) are highly investigated due to being medicinally important targets for drugs. They share certain features with the NPFs, and my introduction presents what is known for the POTs and relate the knowledge to the NPFs. The POTs natural substrate are predominantly di- and –tripeptides with a near limitless variation to the sidechains. In contrast, the NPFs have a more diverse substrate list, and comprise transporters that are highly specific in their substrate preference. This substrates transported by the family will be described in review Article I.

The glucosinolate transporters 1-3 (GTR1-3) belonging to the NPFs, have emerged as a unique model system to investigate the substrate specificity of the NPFs. GTR1 and GTR2 transport glucosinolates without any specific preference, where GTR3 prefers indolic glucosinolates. The substrate specificity difference is hypothesized to lie in the amino acids in the cavity of the transporters, and following a phylogenetic approach, differently conserved amino acids between GTR1/2 and GTR3 were identified to play a role in substrate specificity (Manuscript II).  Amino acids determining transport rate are not as easy to predict. A randomly found point mutation in GTR2 make the transporter hyperactive. In extensive biophysical characterizations, we found the mutation to lead to both changes in transport rate and in several other properties of the transporter (Manuscript III). The synthesis of fluorescent glucosinolates, and the demonstration of transport by the GTRs, lay the foundation for development of a method for high-throughput screening assays for identification of additional amino acids important for substrate specificity and transport rate in the NPFs (Manuscript I). 
The final manuscript describe a method – CRISPR/Cas Prime Editing – for investigations of mutations in planta, which are found to effect properties of the transporters. The manuscript including easy method for cloning of vectors and report of high efficiency prime edits in Arabidopsis thaliana (Manuscript IV).
The results presented in this dissertation provide the groundwork for further studies into the structure-function relationship in the NPFs.

Supervisors
Hussam Hassan Nour El Din Auis, Molecular Plant Biology, PLEN

Assessment committee

Group Leader Christian Loew 
European Molecular Biology Laboratory Hamburg and Centre for Structural Systems Biology, Germany

Group Leader Tonni Grube Andersen 
Max Planck Institute for Plant Breeding Research, Cologne, Germany

Professor Anja Fuglsang, 
Transport Biology, PLEN (chair)